Aspartic proteinases of the A1 family are widely distributed among plant species and have been purified from a variety of tissues. They are most active at acidic pH, are specifically inhibited by pepstatin A and contain two aspartic residues indispensible for catalytic activity. The three-dimensional structure of two plant aspartic proteinases has been determined, sharing significant structural similarity with other known structures of mammalian aspartic proteinases. With a few exceptions, the majority of plant aspartic proteinases identified so far are synthesized with a prepro-domain and subsequently converted to mature two-chain enzymes. A characteristic feature of the majority of plant aspartic proteinase precursors is the presence of an extra protein domain of about 100 amino acids known as the plant-specific insert, which is highly similar both in sequence and structure to saposin-like proteins. This insert is usually removed during processing and is absent from the mature form of the enzyme. Its functions are still unclear but a role in the vacuolar targeting of the precursors has been proposed. The biological role of plant aspartic proteinases is also not completely established. Nevertheless, their involvement in protein processing or degradation under different conditions and in different stages of plant development suggests some functional specialization. Based on the recent findings on the diversity of A1 family members in Arabidopsis thaliana, new questions concerning novel structure-function relationships among plant aspartic proteinases are now starting to be addressed.

Two new aspartic proteinases have been isolated from stigmas of the cardoon Cyncrrn curdunculus L. by a two-step purificalion procedure including extraction at low pH, gel filtration on Superdex 200, and ion-exchange chromatography o n Mono Q. To follow the conventional nomenclature for aspartic proteinases, we have named these proteinases cardosin A and cardosin B. On SDS/PAGE, cardosin A migrated as two bands with apparent molecular masses of 31 000 Da and 15000 Da whercas the chains of cardosin B migrated as bands of 34000 Da and 14000 Da. The partial amino acid sequences of the two cardosins revealed that they are similar but not identical, and that they differ horn the previously reported cardoon proteinases named cynarases, which were assumed to be derived from a common precursor. Although thc cardosins show somc degree of similarity to each other, we could detect no immunological crossreactivity between theni. Both cardosins were active at low pH and were inhibited by pepslatin, with K, values of 3 nM for cardosin A and 1 nM for cardosin B, jndicaring that they belong to the class of aspartic proteinases. Significant differences between thc two enzymes were also found for thc K,,,,/k,,, values for the hydrolysis of two chromophoric synthetic peptides. The active-sitc ionization constants, pK,, and pKC2, for cardosin A are 2.5 -C 0.2 and 5.3 2 0.2, whci-eas for cardosin R they are 3.73 10.09 and 6.7 5 0.1. The results herein described on the structural and kinetic properties of the cardosins indicate that they are the products of distinct genes which havc probably arisen by gene duplication. A scheme for the proteolytic processing of the two enzymes is also proposed.Ke.ywordx: Cynaru curdunculus L. ; aspartyl proteinascs; milk-clotting enzymes; cardosins.Aspartic proteinases are a group of enzymes that share many features in terms of sequence, three-di rnensional structure and catalytic mechanism [I -31. They are widely distributed in nature and have important roles i n biological systems such as precursor protein processing (retroviral proteases), protein degradation (pepsin, cathepsin D and fungal proteases) and blood-pressure regulation (rcnin) (for reviews, see [3-51).Only a small number of aspartic proteinases have been isolated and partially characterised from plants [6-131. These proteinases, in common with most other aspartic proteinascs, havc an acid pH optimum, are inhibited by pepstatin and preferentially cleave pcptide bonds between hydrophobic residues. Little is known about their biological functions, but it has been suggested that plant aspartic proteinases rrre involved in the hydrolysis of storage and intracellular protcins 11 4-171. of about 100 amino acids which bears no sequence similarity with aspartic proteinnses of inamtnalian or microbial origins.The tlowers of cardoon (genus Cynnm) are traditionally used in Portugal for cheese making and their proteinases are among the few eiizymes from vegctal sources that have been used for this purpose. We have previously reported the isolation ...

The flowers ofcardoon (genus Qmara)are traditionally used in Portugal for cheese making. In this work the vegetable rennet of the species Cynam oan/uncu/us L. was characterized in terms of enzymic composition and proteolytic specificity of its proteinases (cardosin A and cardosin B). Cardosin A was found to cleave insulin B chain at the bonds Leul5-Tyrl6, Leul7-Vall8 and Phe2.5-Tyr26. In addition to the bonds mentioned cardosin B cleaves also Glul 3-Ala14, Alal4-Leul5 and Phe24-Phc25 indicating that it has a broader specificity. The kinetic parameters for the hydrolysis of the synthetic peptide Leu-Ser-Phe(NO2)-Nle-Ala-Leu-oMe were also determined and compared to those of chymosin and pepsin. The results obtained indicate that in terms of specificity and kinetic parameters cardosin A is similar to chymosin whereas cardosin B is similar to pepsin. It appears therefore that the enzyme composition of cardoon rennet closely resembles that of calf rennet.

Cork oak (Quercus suber) is native to southwest Europe and northwest Africa where it plays a crucial environmental and economical role. To tackle the cork oak production and industrial challenges, advanced research is imperative but dependent on the availability of a sequenced genome. To address this, we produced the first draft version of the cork oak genome. We followed a de novo assembly strategy based on high-throughput sequence data, which generated a draft genome comprising 23,347 scaffolds and 953.3 Mb in size. A total of 79,752 genes and 83,814 transcripts were predicted, including 33,658 high-confidence genes. An InterPro signature assignment was detected for 69,218 transcripts, which represented 82.6% of the total. Validation studies demonstrated the genome assembly and annotation completeness and highlighted the usefulness of the draft genome for read mapping of high-throughput sequence data generated using different protocols. All data generated is available through the public databases where it was deposited, being therefore ready to use by the academic and industry communities working on cork oak and/or related species.